Hepatitis C virus (HCV) is a hepatotropic virus that mediates a persistent infection and chronic liver disease in millions of people worldwide. HCV persistence is associated with viral strategies to evade innate antiviral immune defenses and a/ interferon (IFN) immune actions that normally limit infection. We have identified HCV genome RNA and the cellular RIG-I protein as the viral pathogen-associated molecular pattern (PAMP) and host factors that trigger innate immunity and IFN production in HCV-infected hepatocytes. Our studies show that in order to evade innate immunity, HCV blocks RIG-I signaling induced by HCV PAMP RNA during the early stages of infection through viral NS3/4A protease cleavage and inactivation of the IPS-1 adaptor protein of the RIG-I pathway. Our research defines the virus/RIG-I pathway interface as a major point of control over HCV infection and immunity. We hypothesize that the processes of viral triggering and control of the RIG-I pathway regulate innate immunity and HCV infection outcome. To investigate this hypothesis we will 1) Determine the nature of the HCV PAMP RNA and signaling mechanisms that trigger the RIG-I pathway and innate immunity in infected cells; 2) Define the domains and cofactor components of IPS-1 that direct innate immune actions and are controlled by the HCV NS3/4A protease; and 3) Define the molecular mechanisms of HCV NS3/4A protease targeting and interaction with IPS-1. Our studies will feature virologic, genetic, biochemical, bioinformatics, and pharmacologic approaches to define the virus-host interactions and therapeutic strategies that regulate innate immunity and HCV infection. Public Health Relevance: Hepatitis C virus (HCV) infects million of people around the world. The virus usually mediates a persistent infection of the liver and is the leading cause of liver disease, liver cancer, and liver transplant. Our work has linked HCV persistence with viral strategies that control innate immunity against infection. The proposed studies will define the virus and host cell processes that trigger and control innate immunity and HCV infection outcome.